Molecular diagnostics aims at the rapid detection of minute amounts of pathogens (typically bacteria) in samples such as blood. Blood is however a complex matrix and comprises white blood cells (leukocytes) for the adaptive immune system, red blood cells (erythrocytes) for oxygen transport, and platelets (thrombocytes) for wound healing. This complicates the direct detection of pathogens in samples such as whole blood, which contain a high amount of cellular material.
Classical detection methods comprise the growth of bacteria on selective media and/or media with indicators. Typically such assays require a cultivation step of at least 1 or 2 days before identification can take place.
For PCR based methods the amount of bacteria in a fresh blood sample is theoretically high enough to be detected without further cultivation of the bacteria present within such sample. However, to allow an early detection of minute amounts of bacteria, large volumes of blood are required. The high amount of DNA in especially white blood cells dramatically increases the background in DNA based detection methods. Also the presence of heme from hemoglobin strongly decreases the activity of DNA polymerase. A microliter of human blood contains about 4,000 to 11,000 white blood cells and about 150,000 to 400,000 platelets. The concentration of DNA in blood is between 30 and 60 μg/ml. It is extremely challenging to detect in a volume of 10 ml of whole blood the presence of about 10 to 100,000 of a bacterial species.
The high amounts of DNA of the white blood cells may give rise to non relevant PCR products, or may scavenge the primers designed for the detection of bacterial DNA. This necessitates a thorough DNA purification and separation of mammalian DNA before the bacterial DNA can be detected via PCR or other methods.
Apart from interfering with the PCR reaction itself the amount of mammalian DNA increases the viscosity of a sample. In addition, proteins and membranes from the lysed mammalian cells form complexes which prevent the filtration of a sample. This is particularly a problem for miniaturized devices. Further dilution of the, already large sample volume, results in unacceptable long manipulation steps.
For the above reasons, methods to remove human DNA from a blood sample are accordingly required.
Methods to specifically assay bacterial DNA in the presence of mammalian DNA are known. Looxter™ from the company SIRSLab uses a method to enrich methylated DNA from a sample. As bacterial DNA is strongly methylated, this approach results in an enrichment of bacterial DNA. Molysis™ from the company Molzym, uses chaotropic agents and detergents to lyse selectively mammalian cells. This lysis step is followed by a digest with a DNAse which is not affected by this chaotropic agent/detergent. Alternative approaches such as commercialized by Roche (Septifast™) rely on PCR primer pairs which are specifically designed to prevent aspecific binding to human DNA and amplification of human DNA.
U.S. Pat. No. 6,803,208 describes a method wherein a highly diluted suspension of blood platelets doped with bacteria is lysed at 37° C. for 15 minutes, whereafter it is possible to filter a small amount of the lysed sample over a 0.4 μm filter for visual inspection of the bacteria which are retained on the filter. This method however does not allow to process large volumes of sample at ambient temperatures.